scholarly journals The effect of salt and site-directed mutations on the iron(III)-binding site of human serum transferrin as probed by EPR spectroscopy

1995 ◽  
Vol 309 (2) ◽  
pp. 403-410 ◽  
Author(s):  
J K Grady ◽  
A B Mason ◽  
R C Woodworth ◽  
N D Chasteen
Keyword(s):  
Binding Site ◽  
Epr Spectroscopy ◽  
Structural Changes ◽  
Hinge Region ◽  
Iron Binding ◽  
Chloride Binding ◽  
Binding Studies ◽  
Directed Mutation ◽  
Binding Isotherms ◽  
Binding Cleft

The effects of site-directed mutation and salt on the iron(III)-binding site of the recombinant half-molecule of the N-terminal lobe (hTf/2N) of human transferrin was studied by EPR spectroscopy. Changes were observed in the EPR spectra of all variants investigated (D63S, D63C, G65R, K206Q, H207E, H249E, H249Q, K296E and K296Q) compared with that of the wild-type protein. The most pronounced changes in the metal site were caused by replacement of the coordinating residues, Asp-63 and His-249, and the non-coordinating residue Lys-296, which is located in the hinge region of the iron-binding cleft. The EPR spectral changes from replacement of other non-coordinating residues were more subtle, indicating small changes in Fe3+ coordination to the protein. The EPR spectrum of variant G65R suggests that it adopts two distinct conformations in solution, one in which the two domains forming the iron-binding cleft are closed and one in which they are open; in the latter instance Asp-63 is no longer coordinated to the Fe3+. Chloride-binding studies on hTf/2N, K206Q, H207E, K296Q and K296E showed similar binding isotherms, indicating that none of the hinge region residues replaced, i.e. Lys-206, His-207 or Lys-296, are the sites of chloride binding. The results show that the coordination environment of the Fe3+ is sensitive to structural changes from site-directed mutation of both remote and coordinated residues and also to chloride-binding and ionic strength effects.

Characterization of the iron binding site in human 5-lipoxygenase by EPR spectroscopy

1993 ◽  
Vol 51 (1-2) ◽  
pp. 177
Author(s):  
N.D. Chasteen ◽  
J.K. Grady ◽  
M.D. Percival ◽  
D. Riendeau
Keyword(s):  
Binding Site ◽  
Epr Spectroscopy ◽  
Iron Binding

scholarly journals Crystal structure ofHalobacterium salinarumhalorhodopsin with a partially depopulated primary chloride-binding site

2016 ◽  
Vol 72 (9) ◽  
pp. 692-699 ◽  
Author(s):  
Madeleine Schreiner ◽  
Ramona Schlesinger ◽  
Joachim Heberle ◽  
Hartmut H. Niemann
Keyword(s):  
Schiff Base ◽  
Binding Site ◽  
Conformational Changes ◽  
Structural Changes ◽  
Chloride Ion ◽  
Chloride Ions ◽  
Halobacterium Salinarum ◽  
Chloride Binding ◽  
High Ph ◽  
Chloride Depletion

The transmembrane pump halorhodopsin in halophilic archaea translocates chloride ions from the extracellular to the cytoplasmic side upon illumination. In the ground state a tightly bound chloride ion occupies the primary chloride-binding site (CBS I) close to the protonated Schiff base that links the retinal chromophore to the protein. The light-triggeredtrans–cisisomerization of retinal causes structural changes in the protein associated with movement of the chloride ion. In reverse, chemical depletion of CBS I inNatronomonas pharaonishalorhodopsin (NpHR) through deprotonation of the Schiff base results in conformational changes of the protein: a state thought to mimic late stages of the photocycle. Here, crystals ofHalobacterium salinarumhalorhodopsin (HsHR) were soaked at high pH to provoke deprotonation of the Schiff base and loss of chloride. The crystals changed colour from purple to yellow and the occupancy of CBS I was reduced from 1 to about 0.5. In contrast toNpHR, this chloride depletion did not cause substantial conformational changes in the protein. Nevertheless, two observations indicate that chloride depletion could eventually result in structural changes similar to those found inNpHR. Firstly, the partially chloride-depleted form ofHsHR has increased normalizedBfactors in the region of helix C that is close to CBS I and changes its conformation inNpHR. Secondly, prolonged soaking ofHsHR crystals at high pH resulted in loss of diffraction. In conclusion, the conformation of the chloride-free protein may not be compatible with this crystal form ofHsHR despite a packing arrangement that hardly restrains helices E and F that presumably move during ion transport.

scholarly journals Protein Sensing Device with Multi-Recognition Ability Composed of Self-Organized Glycopeptide Bundle

2020 ◽  
Vol 22 (1) ◽  
pp. 366
Author(s):  
Mao Arai ◽  
Tomohiro Miura ◽  
Yuriko Ito ◽  
Takatoshi Kinoshita ◽  
Masahiro Higuchi
Keyword(s):  
Binding Site ◽  
Lipid Bilayer ◽  
Structural Changes ◽  
Bilayer Membrane ◽  
Lipid Bilayer Membrane ◽  
Target Protein ◽  
Self Organization ◽  
Specific Response ◽  
Target Proteins ◽  
Recognition Ability

We designed and synthesized amphiphilic glycopeptides with glucose or galactose at the C-terminals. We observed the protein-induced structural changes of the amphiphilic glycopeptide assembly in the lipid bilayer membrane using transmission electron microscopy (TEM) and Fourier transform infrared reflection-absorption spectra (FTIR-RAS) measurements. The glycopeptides re-arranged to form a bundle that acted as an ion channel due to the interaction among the target protein and the terminal sugar groups of the glycopeptides. The bundle in the lipid bilayer membrane was fixed on a gold-deposited quartz crystal microbalance (QCM) electrode by the membrane fusion method. The protein-induced re-arrangement of the terminal sugar groups formed a binding site that acted as a receptor, and the re-binding of the target protein to the binding site induced the closing of the channel. We monitored the detection of target proteins by the changes of the electrochemical properties of the membrane. The response current of the membrane induced by the target protein recognition was expressed by an equivalent circuit consisting of resistors and capacitors when a triangular voltage was applied. We used peanut lectin (PNA) and concanavalin A (ConA) as target proteins. The sensing membrane induced by PNA shows the specific response to PNA, and the ConA-induced membrane responded selectively to ConA. Furthermore, PNA-induced sensing membranes showed relatively low recognition ability for lectin from Ricinus Agglutinin (RCA120) and mushroom lectin (ABA), which have galactose binding sites. The protein-induced self-organization formed the spatial arrangement of the sugar chains specific to the binding site of the target protein. These findings demonstrate the possibility of fabricating a sensing device with multi-recognition ability that can recognize proteins even if the structure is unknown, by the protein-induced self-organization process.

scholarly journals Interbase-FRET binding assay for pre-microRNAs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mattias Bood ◽  
Anna Wypijewska del Nogal ◽  
Jesper R. Nilsson ◽  
Fredrik Edfeldt ◽  
Anders Dahlén ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Drug Targets ◽  
Structural Changes ◽  
Small Sample Size ◽  
Resonance Energy ◽  
Small Sample ◽  
Titration Calorimetry ◽  
Drug Candidate ◽  
Binding Studies ◽  
Aberrant Expression

AbstractThe aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA–target binding studies.

scholarly journals Engineered occluded apo-intermediate of LacY

2018 ◽  
Vol 115 (50) ◽  
pp. 12716-12721 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
H. Ronald Kaback
Keyword(s):  
Binding Site ◽  
Lactose Permease ◽  
Binding Studies ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Transport Cycle ◽  
Open Structures ◽  
Unrestricted Access ◽  
Tight Association ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

Identification of the chloride-binding site in the human red and green color vision pigments

Biochemistry ◽  
1993 ◽  
Vol 32 (9) ◽  
pp. 2125-2130 ◽  
Author(s):  
Zhiyan Wang ◽  
Ana B. Asenjo ◽  
Daniel D. Oprian
Keyword(s):  
Binding Site ◽  
Color Vision ◽  
Chloride Binding ◽  
Green Color
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